Physiological Genomics

Mice housed at room temperature (RT, 25{degrees}C) experience chronic mild cold stress compared with those housed at thermoneutrality (TN, 30{degrees}C). We hypothesized that cold stress suppresses circadian transcript expression in peripheral tissues. RNA-seq of hearts, livers, and diaphragms collected every 4 hours over 48 hours in constant darkness identified mRNA transcripts exhibiting {approx}24-hour rhythms (REGs). TN produced tissue-specific changes in REG number, identity, and phase without altering core circadian clock transcript levels. Cardiac REGs increased 4-fold, diaphragm REGs 1.5-fold, and hepatic REG identity shifted substantially. GO analysis revealed coordinated reorganization of rhythmic metabolic programs in the heart and liver. These data demonstrate that ambient housing temperature has tissue-specific effects on the number, identity, and temporal organization of rhythmically expressed transcripts in the heart, liver, and diaphragm.

Circadian rhythms regulate diverse physiological processes, including metabolism, and their disruption has been implicated in metabolic disorders such as obesity. However, the tissue-specific effects of obesity on peripheral circadian clocks remain incompletely understood. Here, we investigated the impact of high-fat diet (HFD)-induced obesity on circadian gene expression in skeletal muscle, liver, and white adipose tissue (WAT). Mice were fed either a regular diet (RD) or HFD for 6 weeks, followed by tissue collection at 4-hour intervals over a 24-hour period. Under RD conditions, key circadian regulators and their downstream targets exhibited robust 24-hour oscillations across all tissues. In contrast, HFD feeding induced distinct, tissue-specific alterations. In the liver, Per2, Dbp, and Rev-erb showed phase-advanced expression patterns, whereas in WAT, rhythmic expression was markedly attenuated. Notably, skeletal muscle largely preserved circadian gene expression patterns, indicating relative resistance to HFD-induced circadian disruption. In addition, HFD feeding altered metabolic gene expression in adipose tissue, characterized by reduced Pgc1 expression and increased Leptin expression. Together, these findings demonstrate that HFD-induced obesity differentially disrupts peripheral circadian clocks in a tissue-specific manner and highlight skeletal muscle as a relatively resilient tissue. These results provide insight into how circadian dysregulation contributes to metabolic abnormalities in obesity.

Objective: To characterize genome-wide DNA methylation patterns associated with sepsis using the Infinium Methylation EPIC v2.0 platform and to evaluate the feasibility of pooled methylation profiling in a pilot critical care cohort. Design: Single-center pilot epigenome-wide association study using pooled whole-blood genomic DNA and pool-level bioinformatic analysis. Setting: Academic medical center. Patients: Fifty critically ill adults enrolled within 48 hours of illness onset and 20 healthy controls. Interventions: None. Measurements and Main Results: Critically ill patients required mechanical ventilation and/or vasopressor support. Sepsis was defined according to Sepsis-3 criteria. Seventy individual samples were organized into 14 intended pools of 5 individuals each: 7 sepsis pools, 3 critically ill non-septic pools, and 4 healthy-control pools. One critically ill non-septic pool was excluded because of poor DNA quality, yielding 13 analyzable pools. For the primary pooled comparison, 7 sepsis pools were compared with 6 non-sepsis comparator pools comprising 2 critically ill non-septic and 4 healthy-control pools. After quality control and preprocessing with SeSAMe, 876,094 CpG sites were retained. The initial pool-level screen identified 170,897 candidate differentially methylated regions. Application of stringent secondary filters (false discovery rate <= 1%, absolute delta-beta >= 7.5%, and >= 5 CpGs per region) yielded a high-confidence subset with marked directional skewing, including 155 hypomethylated and 32 hypermethylated regions in sepsis. Differentially methylated region-associated genes were enriched in myeloid leukocyte activation, myeloid leukocyte-mediated immunity, defense response to bacterium, neutrophil granule biology, and hematopoietic cell lineage pathways. Additional signals involved microRNA-associated targets, ribosome biogenesis, RNA processing, long noncoding RNAs, and previously uncharacterized loci. Conclusions: In this pilot pooled EPIC v2.0 study, sepsis was associated with a biologically coherent, predominantly hypomethylated methylation signature enriched in myeloid and host-defense pathways. These findings support the feasibility of pooled methylation profiling for discovery-oriented sepsis biobank studies but should be interpreted as hypothesis-generating given the pool-level design, limited effective sample size, heterogeneous comparator group, and lack of direct validation against individual-level methylation profiles.

Zinc is an essential trace element involved in numerous biological processes, including cellular signalling, development, and reproduction. Zinc homeostasis is regulated by zinc transporters, yet the physiological roles of many transporters remain poorly understood in vivo. Here, we investigated the function of the zinc transporter ZIP9 (SLC39A9) using a zebrafish (Danio rerio) knockout model. Elemental imaging using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) revealed altered zinc distribution in zip9-deficient larvae. Synchrotron-based X-ray fluorescence (XRF) imaging further showed reduced zinc levels in the brain region of mutant zebrafish. Consistent with these observations, loss of zip9 was associated with altered expression of key neuroendocrine genes within the hypothalamic-pituitary-gonadal (HPG) axis. Zip9 mutant females exhibited disrupted ovarian follicle development, reduced spawning rates, and decreased egg production. In addition, embryos derived from zip9 mutant parents displayed reduced size, impaired early development, and decreased survival. Together, these findings identify ZIP9 as a regulator of zinc distribution in vivo and suggest that ZIP9-mediated zinc signalling contributes to reproductive regulation in zebrafish.

We investigated effects of three aerobic exercise interventions, varying in amount and intensity with durations of 8-9-months on small RNA (smRNA) expression and regulatory pathways in skeletal muscle and plasma from 120 participants. Using untargeted smRNA sequencing focused on miRNAs and piRNAs, adjusting for demographics and bodyweight, we identified 124 muscle smRNAs altered by exercise amount and 15 by intensity, and 47 plasma smRNAs altered by intensity and one by amount. These smRNAs were enriched in metabolic, transcriptional, translational, and cell cycle pathways. Exercise-induced changes in several smRNAs-six from muscle and five from plasma-and exercise-induced reduction in body weight, aligned with improvement in insulin sensitivity (p<0.05). These findings demonstrate tissue-specific regulation of smRNAs by exercise and identify potential candidates for exercise mimetics to modulate muscle insulin sensitivity.

2.Childhood atopic disease is linked to delayed gut microbiome development and metabolic dysfunction, however microbial drivers remain unclear. To explore microbial correlates of asthma risk during a time of active gut microbiome development, we analyzed stool from 6-month-old infants at high asthma risk (HR) or healthy controls (HC), using Genome-resolved metagenomics (HR=7; HC=12) and untargeted metabolomics (HR=11; HC=15). We recovered 82 bacterial species-level metagenomic-assembled genomes (MAGs). Global Taxonomic composition did not differ by asthma risk. Anticipating that key differences might associate with specific genomes, a machine-learning approach pinpointed Bacteroides cellulosilyticus, Hungatella effluvii, and Enterocloster aldenensis as linked with asthma risk status. All three species were more abundant in HC infants and the B. cellulosilyticus genome was enriched for carbohydrate metabolism genes relative to other MAGs. Metabolomic profiling revealed variance associated with asthma risk (PERMANOVA, R2 =0.069, p=0.016). HR fecal metabolomes were enriched in simple sugars, whereas HC contained more nitrogenous compounds. Integrative genome-metabolic modeling of compounds that significantly differentiate asthma-risk groups revealed risk-dependent interactions with community-encoded metabolic potential (CEP), for arabinose and agmatine, whose fecal concentrations are linked with B. cellulosilyticus and H. effluvii functional traits respectively. These findings suggest that microbial-influenced metabolic differences associate with asthma risk at 6 months, with B. cellulosilyticus and H. effluvii emerging as candidate bacteria influencing this observed metabolic remodeling. 3. Impact statementLeveraging a random forest classifier, we identified three bacterial species (Bacteroides cellulosilyticus, Hungatella effluvii, and Enterocloster aldenensis) as distinguishing features enriched in healthy 6-month old infant microbiomes compared to those at high risk of asthma development (HR). We developed an approach to integrate metabolomics and metagenomic-derived microbiome community encoded potential (CEP) with clinical outcomes to identify fecal metabolites whose concentrations are likely to be influenced by the microbiome. Fecal arabinose concentrations were positively associated with CEP in healthy infants, but not in HR subjects who exhibited elevated concentrations irrespective of CEP. These data implicate microbial activity as a contributor to the concentration of this metabolite in healthy but not HR infants. With a leave-one-out-cross-validation, we identified B. cellulosilyticus as a contributor to fecal arabinose concentrations. Our data indicate that microbial functional deficits in HR infants is associated with altered gut metabolic dysfunction during microbiome maturation. 4. Data summaryDurack et. al [1] is the source of the metabolomics data utilized in this study. The authors confirm that all other supporting data, code and protocols have been provided within the article or through supplementary data files.

Gene-by-environment (GxE) interactions play a major role in shaping both phenotypic and molecular variation, with important implications for human health and disease. In this study, we used the Doxycycline (Dox) regulated, tetracycline-responsive (Tet-Off) promoter system to sequentially reduce or titrate gene expression levels of the essential yeast transcription factor Repressor Activator Protein 1 (RAP1) similar to a hypomorph allele series, across three distinct environments: Yeast Peptone Dextrose (YPD) media, YPD media with Heat Shock (HS), and Yeast Peptone Acetate (YPAC) media. We then performed RNA sequencing (RNA Seq) to assess global transcriptional responses to RAP1 reduction in these different growth environments. Our analysis first focused on the independent effects of varying RAP1 expression levels within and across environments. We then explored GxE interactions, revealing a subset of genes with significant consequences of reduced levels of RAP1 and environment-specific expression patterns. Notably, many genes exhibited opposite effects of RAP1 titration on gene expression when yeast were grown in YPAC media compared to YPD media and/or HS, suggesting environment-dependent regulatory architecture. This design reveals how cells integrate internal transcriptional and regulatory changes with external environmental cues, providing a deeper view of GxE architecture. Using Weighted Gene Co-expression Network Analysis (WGCNA), we identified co-regulated gene modules, and by combining this with transcription factor motif enrichment tests, our study identified candidate regulators driving their dynamics. Our findings demonstrate that gene regulatory networks can vary dramatically depending on the environmental context an organism experiences, which can then influence the specific phenotypes produced by a particular genetic perturbation. This illustrates the complexity of genotype-environment interactions and the importance of studying gene function in multiple environments to gain a truly comprehensive understanding of a genes sometimes numerous and diverse functions.

Of the three dietary macronutrients, protein plays an especially pivotal role in physiological functions. Nevertheless, the behavioural control of protein intake is poorly understood. In this study, we used Feeding Experimentation Devices (FED3s) to examine the structure of ingestive behaviour in mice given access to diets varying in protein content. Adult C57BL/6NRj mice were contact-housed in pairs in custom-made cages with perforated dividers, each having access to an individual FED3 unit. Mice were given ad libitum access to either 20 mg control, non-restricted (NR) pellets (20% casein) or 20 mg protein-restricted (PR) pellets (5% casein) from FED3s on free-feeding mode. Each pellet retrieval event was timestamped ~24 h/day. All mice experienced both diets for 7 days with order of diet presentation counterbalanced (i.e., NR[-&gt;]PR and PR[-&gt;]NR). Analysis of dynamics of pellet intake per day revealed that mice that were initially protein-restricted first showed a decrease in pellet intake before increasing on later days and exhibiting a persistent high level of intake once non-restricted diet was available. The group that was initially non-restricted exhibited a blunted response to the same diet manipulation. In addition, we clustered pellet retrieval data into discrete clusters of feeding events and used a mathematical approach to determine the boundary of meals (2-5 pellets), separated from "snacks" (1 pellet) and "feasts" (>5 pellets). We identified alterations in meal patterning in response to diet manipulation with protein restriction increasing "snacking" and leading to increased meal number, and reduced meal size. Moreover, restored access to NR diet, elicited "feasting". These effects depended on the sequence of diets the mice experienced, such that the effects were stronger in initially protein restricted mice compared to those initially non-restricted. In summary, our findings show that manipulation of dietary protein levels affects meal patterning in adult mice.

BackgroundMetabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent pediatric disorder with limited treatment options, primarily due to an incomplete understanding of its molecular drivers. Recent research underscores the role of microbial guilds in metabolic health, but the mechanisms by which dysbiosis driven by core species and co-abundant symbionts disrupt metabolic homeostasis in pediatric MASLD remain unclear. ResultsHere, we conducted integrated metagenomic and metabolomic analyses on 285 pediatric subjects including MASLD patients, obese and healthy controls. The gut dysbiosis in MASLD was characterized by a depletion of Phocaeicola vulgatus, Bacteroides uniformis, Parabacteroides distasonis, and Bacteroides thetaiotaomicron. Co-abundance network analysis, integrating our cohort with four public datasets, identified these species as core guild members associated with MASLD. Microbial enrichment analysis showed significant disruptions in carbohydrate metabolism, particularly the downregulation of the tricarboxylic acid (TCA) cycle, fructose and sucrose metabolism, and pentose and glucuronate interconversions. P. vulgatus and B. uniformis were identified as dominant species linked to the downregulation of KEGG orthologs (KOs) in these disrupted pathways that were inversely correlated with hepatic injury biomarkers. CAZyme database analysis further emphasized P. vulgatus as the primary contributor to glycoside hydrolases involved in monosaccharide utilization. Finally, both untargeted and targeted metabolomics analysis validated a disrupted metabolic network centered on the TCA cycle and monosaccharide metabolism in pediatric MASLD. ConclusionOur findings suggest the core guild species P. vulgatus and B. uniformis may serve as critical regulators of carbohydrate metabolism in pediatric MASLD, offering potential mechanistic targets for gut microbiome-based interventions.

Study questionDoes the human placenta utilize the creatine phosphagen system for energy homeostasis during development? Summary answerComponents of the creatine (Cr)-creatine kinase (CK)-phosphocreatine (PCr) system are dynamically expressed by the trophoblast and mesenchymal compartments throughout gestation wherein creatine kinase is required for cellular ATP metabolism, cell cycle, and proliferation of trophoblast cells. What is known alreadyThe Cr-CK-PCr system maintains ATP homeostasis in tissues with high energy demand and is required for proliferation, migration, and invasion of tumor cells. The term human placenta can synthesize and transport creatine locally. Early placental development involves trophoblast proliferation, an event requiring ATP, but the role of the creatine phosphagen system during early placental development remains unknown. Study design, size, durationWe performed immunohistochemistry (IHC) and immunofluorescence (IF) for different components (biosynthesis, transport, utilization) of the Cr-Ck-PCr system in human placentae (n=3/group) across gestation including first trimester, second trimester, and term. Using primary human trophoblast stem cells (hTSCs) and trophoblast organoids (TO), we determined the role of the creatine phosphagen system in trophoblast growth by functional inhibition of creatine kinase. Participants/materials, setting, methodsIHC/IF were performed in human placentae across gestation for proteins involved in biosynthesis (AGAT and GAMT), transport (SLC6A8, SLC22A15, and SLC6A13) and utilization (CKB and CKMT1) of creatine to determine the presence of the creatine phosphagen system locally in the placenta. For delineating the functional importance of this system in placental development, cyclocreatine (cCr), a creatine analogue, was used for functional inhibition of CK. Primary hTSCs were culture in medium containing 0 (control), 1, 10, 20 mM cCr for 48 hours followed by analysis of cell growth (cell count), cell cycle (EdU incorporation assay), apoptosis (Annexin V/PI flow cytometry), energy metabolism (Sea horse mito-stress and glycolytic stress tests), and gene expression (qPCR). Primary TO were also treated with 20mM cCr for 6 days in vitro to determine the role of Cr-CK-PCr system in placental development. Main results and the role of chanceAGAT localized to the fetal villous mesenchyme, while GAMT was broadly expressed in the trophoblast and fetal mesenchyme compartments across gestation. CKB localized primarily to fetal mesenchyme with strongest expression at term. CKMT1 was broadly expressed in all trophoblast subtypes. SLC6A8 was abundant in early syncytiotrophoblast but absent at term, where its expression shifted to fetal blood vessels. SLC22A15 was expressed in the endothelial cells of fetal capillaries across gestation. In primary hTSCs, cyclocreatine (20mM) treatment reduced proliferation (P<0.001), decreased expression of trophoblast epithelial marker EGFR (P<0.05), induced G0/G1 and G2/M arrests (P<0.0001), enhanced early and late apoptosis (P<0.0001), and downregulated GPX8 expression (P<0.05). Seahorse analysis revealed marked reductions (P<0.01) in mitochondrial (basal, maximal, and ATP-linked) and glycolytic (rate, capacity, and reserve) function compared to controls. In primary human TO, cyclocreatine treatment reduced the growth of organoids (P<0.05) as well the expression of EGFR (P<0.05). Large scale dataN/A Limitations, reasons for cautionFurther experiments assessing apoptosis, cellular stress and redox imbalance may provide more mechanistic role of the creatine phosphagen system in trophoblast metabolism and function. Since the functional role of the Cr-CK-PCr system was investigated in vitro, findings of this study should be taken with caution for implications of in vivo placental development. Nevertheless, reproducible results of reduced growth of trophoblast cells using both 2D and 3D cultures is highly suggestive of the importance of the creatine phosphagen system in early placental development. Wider implications of the findingsThis study provides foundational knowledge that the placenta contains the creatine phosphagen system, known for ATP homeostasis, and that this system ensures proper cell division, survival and placental development. Dysregulation of components of Cr-CK-PCr system in placenta has been observed in pregnancy disorders such as preeclampsia and fetal growth restriction warranting continued investigation into mechanisms and potential remediation using creatine supplementation. Stem cells share similar metabolic features so findings of this study can be implicated in other stem cells models as well. Study funding/competing interest(s)This work was supported by CIRM EDUC4-12804 Interdisciplinary Stem Cell Training Grant and a Lalor Foundation Postdoctoral Fellowship awarded to NS, and by the California Institute for Regenerative Medicine (DISC0-13757) and the National Institute of Child Health and Human Development (R01-HD096260) award to FS. The authors have no competing interest to declare.

Background: Post-operative hypotension and vasoplegia are well recognised following cardiac surgery but remain poorly characterised after major non-cardiac surgery, despite associations with acute kidney injury (AKI), cardiovascular complications, and increased mortality. Dysregulation of the renin angiotensin aldosterone system (RAAS) may underpin haemodynamic instability in this setting, yet data in abdominal surgery are limited. Objectives: The POLECAT (Perioperative delta Renin) study aims to determine whether changes in circulating renin concentration (delta renin) from pre-operative baseline to the early post-operative period are associated with post-operative vasoplegia in patients undergoing major abdominal surgery requiring intensive care admission. Methods: POLECAT is a single-centre, prospective observational study conducted at a UK tertiary referral hospital. Adult patients undergoing planned or emergency abdominopelvic surgery with anticipated intensive care admission are enrolled. Blood samples are obtained pre-operatively, within four hours post-operatively, and on post-operative day one to measure renin and a panel of endothelial, renal, and immune biomarkers. The primary outcome is post-operative vasoplegia, defined as the requirement for a vasopressor infusion at 08:00 on post-operative day one. Secondary outcomes include alternative vasoplegia definitions, AKI (KDIGO criteria), vasopressor burden, organ dysfunction, cardiovascular complications, length of stay, and mortality. Multivariable regression, receiver operating characteristic analyses, and predefined subgroup analyses will be performed, with sensitivity analyses addressing missing data. Conclusions: This study will clarify the relationship between peri-operative RAAS dysfunction and vasoplegia following major abdominal surgery. Findings may support biomarker-guided risk stratification and inform future interventional trials targeting haemodynamic instability in this high-risk population.

Metatranscriptomic (MTX) sequencing enables profiling of gene expression across microbial communities, providing a framework for linking genetic potential with functional activity. However, standard pipelines report normalized abundances rather than raw counts, limiting the use of count-based RNA-seq methods, while Gaussian-based alternatives rely on transformations and assumptions that are often poorly suited to MTX data. We propose a new modeling framework for differential expression analysis of MTX data, built on a scale mixture of exponential distributions, that incorporates DNA abundance to adjust for genomic potential, accommodates subject-specific random effects, treats zeros as left-censored, and employs a mixture prior to handle extreme sparsity. Applied to the IBDMDB multi-omics cohort, differential expression results vary substantially across models, including among Gaussian approaches with different pseudocount choices. Our approach identifies a distinct subset of candidate genes not detected by existing Gaussian methods; these may provide useful leads toward a novel understanding of transcriptomic patterns associated with dysbiosis in inflammatory bowel disease. Estimated dysbiosis effect directions are consistent between our model and Gaussian-based approaches, while effect sizes from our model tend to be larger in absolute value.

Ramadan fasting represents a natural model of prolonged daily intermittent fasting associated with metabolic and circadian alterations. This study investigated longitudinal changes in intracortical excitability across pre-, mid-, and post-Ramadan timepoints in healthy adults observing Ramadan fasting. Thirty fasting participants underwent paired-pulse transcranial magnetic stimulation at three timepoints (pre-, mid-, and post-Ramadan). A non-fasting control group (n = 11) was assessed at pre- and mid-Ramadan. Conditioned motor-evoked potentials were recorded at interstimulus intervals of 2-10 ms and normalized to unconditioned responses. A linear mixed-effects model assessed effects of Timepoint and interstimulus interval (ISI). Secondary outcomes included blood glucose, cognitive performance, sleep duration, and reaction time. A significant main effect of Timepoint (p < 0.001) indicated longitudinal modulation of intracortical excitability, with increased MEP ratios at mid-Ramadan and partial persistence post-Ramadan. The ISI effect confirmed the inhibition-facilitation gradient (p < 0.001). The Timepoint x ISI interaction was not significant (p = 0.566), indicating a global shift in excitability without ISI-specific modulation. Blood glucose and sleep duration decreased significantly at mid-Ramadan. Ramadan fasting is associated with a time-dependent increase in intracortical excitability, most appropriately interpreted as a generalized shift rather than selective modulation of inhibitory or facilitatory circuits. These changes occur in the context of concurrent metabolic and sleep alterations and may reflect combined influences of fasting-related metabolic state and reduced sleep duration; however, these factors cannot be disentangled within the present design.

Astrocytic homeostatic programs, many of which are regulated by the circadian clock, are disrupted early in neurodegenerative disease. The core clock transcription factor (TF) BMAL1 is required for normal astrocyte function, but its role during disease remains unclear. This is partly because methods for identifying cell type-specific TF binding sites are limited. Here, we developed MACS-Calling Cards (MACS-CC), a strategy for mapping astrocyte-specific TF occupancy in vivo. We used MACS-CC to define BMAL1 binding in the Cln3{Delta}ex7/8 mouse model of CLN3 disease, a fatal neurodegenerative disorder marked by early astrocyte dysfunction and circadian disruption. BMAL1 binding was extensively redistributed in Cln3{Delta}ex7/8 astrocytes: wild-type-specific binding sites enriched near glial differentiation genes, whereas Cln3{Delta}ex7/8-specific sites lacked functional enrichment. Consistent with these changes, Cln3{Delta}ex7/8 astrocytes decreased expression of mature astrocyte markers. To define mechanisms underlying BMAL1 retargeting, we tested whether altered chromatin accessibility explained the changes in BMAL1 binding. Although chromatin accessibility was broadly remodeled, differential accessibility did not predict BMAL1 redistribution. Instead, motif analysis suggested that loss of cooperative TF interactions drives BMAL1 retargeting. These findings demonstrate that MACS-CC enables astrocyte-specific TF occupancy mapping and reveals mechanisms behind early rewiring of circadian regulatory programs within a model of a neurodegenerative disease. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/721783v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1ada239org.highwire.dtl.DTLVardef@7564a3org.highwire.dtl.DTLVardef@122222forg.highwire.dtl.DTLVardef@1f2729c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Haemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening hyperinflammatory syndrome characterised by uncontrolled immune activation. Reduced high- and low-density lipoprotein cholesterol and hypertriglyceridaemia are reported in HLH, suggesting lipid metabolism disturbances although in-depth serum metabolomic analysis is lacking in HLH. Here a lipid-focused NMR spectroscopy platform was used to define the serum metabolomic landscape of adults hospitalised with HLH compared to adults with sepsis (HLH-mimic) and rheumatic disease (potential HLH drivers/triggers), following surgical resection of solid organ cancer (non-infectious acute inflammation controls) and healthy controls (HCs). Serum metabolites distinguished HLH from HCs with high accuracy (>91.36%) using multiple machine learning models. The top classifying features included elevated apolipoprotein-B (ApoB)-containing low, intermediate, and very low-density lipoprotein particles; and lipoprotein remodelling towards triglyceride enrichment and cholesterol depletion. Differentially abundant metabolites in HLH compared to all control groups were enriched in pathways related to lipid metabolism including: 'Lipid particles composition', 'Plasma lipoprotein clearance', 'Plasma lipoprotein remodelling', 'Glucose homeostasis' and 'Amino acid metabolism'. Metabolomic results were validated using matched whole blood RNA-sequencing which identified differentially expressed genes enriched in metabolic modules associated with lipid, amino acid, and glucose metabolism, supporting a coordinated metabolic dysregulation in HLH from a transcriptomic to metabolomic level. Finally, twenty-seven metabolites including ApoB-containing, triglyceride-rich lipoproteins and saturated fatty acids distinguished HLH from all disease controls (AUC>0.70) either alone or combined as a metabolomic signature. Elevated ApoB and ApoB:ApoA1 ratio in HLH vs sepsis and HCs were validated by ELISA, supporting their utility as biomarkers to distinguish HLH from other hyperinflammatory syndromes.

BackgroundNeonatal sepsis is a major cause of infant morbidity and mortality worldwide, particularly in preterm and very low birthweight babies. Fundamental differences between neonates and adults warrant clinically relevant models of neonatal sepsis. Here, we describe a preclinical fecal-slurry (FS)-induced peritonitis model of polymicrobial sepsis in neonatal rats, along with a novel neonatal rat sepsis score (nRSS) to monitor illness severity. MethodsPeritonitis was induced in 3-day-old Sprague Dawley rats by intraperitoneal injection of various doses (0.3-1.5mg/g body weight) of fecal slurry (FS); control pups received equivalent doses of vehicle. All pups received analgesics (buprenorphine), antibiotics (ampicillin and gentamicin), and fluids (saline) to model clinical standards of sepsis treatment. Time-dependent changes in circulating cytokines (IL-6, IL-1{beta}) and biomarkers of sepsis pathology (hemoglobin, glucose, alanine transaminase [ALT] levels) were assessed and correlated with nRSS scores. ResultsFS administration caused a dose-dependent increase in severity of sepsis over time, as indicated by increases in mortality rates (based on predefined criteria for euthanasia), nRSS scores, as well as time-dependent changes in circulating glucose, hemoglobin, IL-6, IL-1{beta}, and ALT activity levels. nRSS scores correlated with all quantitative measures of sepsis pathology. Notably, females showed higher mortality and higher early NRSS scores than males at moderate to high FS doses, yet biochemical markers and time of death did not differ between sexes, suggesting that the apparent female vulnerability may reflect more conspicuous behavioral manifestations of illness rather than greater underlying physiological severity. ConclusionInduction of peritonitis in rats at postnatal day 3 produced a consistent and reproducible model of polymicrobial neonatal sepsis. Illness severity was monitored using a newly developed nRSS. By minimizing distress and incorporating standards of care, this model and scoring system may serve as a platform for future investigations into the underlying mechanisms and potential therapeutic interventions for neonatal sepsis. ImpactO_LIA clinically relevant rat model of neonatal polymicrobial sepsis was developed, incorporating standards of care (analgesics, antibiotics, and fluid resuscitation) to better reflect the clinical context in which preclinical findings must ultimately translate. C_LIO_LIA novel neonatal rat sepsis scoring system (nRSS) was developed and validated, providing a sensitive, non-invasive measure of disease severity that correlates with biochemical markers and predicts mortality. C_LIO_LIFemale pups showed higher mortality and earlier behavioral signs of illness than males despite equivalent biochemistry, highlighting that clinical scores may capture sex-dependent vulnerability not apparent in standard biochemical measures. C_LIO_LITogether, this model and scoring system offer a refined platform for mechanistic and therapeutic studies of neonatal sepsis while advancing the welfare-conscious 3Rs principles essential to rigorous preclinical research C_LI

Perinatal opioid exposure is a prevalent clinical concern linked to respiratory instability and adverse infant outcomes. The opioid buprenorphine is prescribed as a medication for opioid use disorder during pregnancy and used to treat neonatal opioid withdrawal syndrome, yet its direct effects on neonatal control of breathing have not been examined. Here, we asked how acute buprenorphine exposure affects breathing at rest, and during chemoreceptor stimulation. Using dual-chamber head-out plethysmography, we measured pulmonary ventilation rate ([V]I) and metabolic rate in awake male and female Sprague-Dawley neonatal rats on postnatal days 4-5 (P4-5) during eupnea and a hypoxic-hypercapnic (HH) challenge. The effects of buprenorphine and two opioid receptor antagonists, naloxone hydrochloride, or peripherally restricted naloxone methiodide, were assessed using a repeated measures design. [V]I during eupnea and HH were markedly depressed following buprenorphine administration. Buprenorphine reduced [V]O2 and [V]CO2 and produced ventilatory equivalents for O2 and CO2 consistent with frank hypoventilation, driven by reduced breathing frequency and tidal volume (VT). When administered after buprenorphine, neither naloxone hydrochloride nor naloxone methiodide could rescue the buprenorphine-mediated hypoventilation in eupnea or during HH. In contrast, pre-treatment with either naloxone hydrochloride or naloxone methiodide attenuated buprenorphine-induced hypoventilation by preserving VT. These findings demonstrate that neonatal protective chemoreceptor reflexes are depressed by buprenorphine and suggest that pre-treatment with a peripheral opioid receptor antagonist could mitigate buprenorphine-induced hypoventilation without inducing opioid withdrawal. Key PointsO_LIAcute buprenorphine exposure significantly depressed pulmonary ventilation rate ([V]I) during eupnea and hypoxic hypercapnia (HH) in awake neonatal rats. C_LIO_LIBuprenorphine-induced hypoventilation was driven by reduced tidal volume (VT) and breathing frequency. C_LIO_LIBuprenorphine also reduced oxygen consumption ([V]O2) and carbon dioxide production ([V]CO2). C_LIO_LINaloxone given after buprenorphine failed to reverse hypoventilation. C_LIO_LIIn contrast, pre-treatment with either naloxone hydrochloride or peripherally restricted naloxone methiodide mitigated buprenorphine-induced hypoventilation by preserving VT. C_LI

BackgroundInflammation-induced pancreatic islet-cell death and dysfunction are key aspects of both type 1 and type 2 diabetes. Stem cell-derived islets (SC-islets) are an emerging tool in diabetes research, however, our understanding of how inflammation affects SC-islet function is incomplete. We therefore aimed to thoroughly characterize how SC-islets respond to pro-inflammatory cytokines at the functional and transcriptomic levels in comparison with human primary islets and EndoC-{beta}H5 cells. MethodA 7-stage differentiation protocol was used to generate SC-islets with insulin-, glucagon-, and somatostatin-positive cells. SC-islets, primary human islets and EndoC-{beta}H5 cells were exposed to different doses of pro-inflammatory cytokines (IL-1{beta} + IFN{gamma} + TNF) including a high dose for up to 48 h and a low dose up to 144 h to mimic the intense islet inflammation in T1D and chronic low-grade inflammation in T2D, respectively. Differential gene expression (RNA-seq), cell death, activation of key signalling proteins, hormones, and chemokine secretion were determined. ResultsBasal expression of key islet-cell identity genes in SC-islets correlated well with that of primary islets and EndoC-{beta}H5 cells. In SC islets, cytokines dose-dependently induced activation of key proximal signalling pathways (NF{kappa}B, STAT1, and JNK), upregulation of major histocompatibility complex (MHC) class I, and increased cell death (cytotoxicity and caspase 3/7 activity). In head-to-head experiments, SC-islets displayed similar cytokine responses particularly as primary islets regarding induction of cell death, chemokine secretion, differential gene expression, and protein levels of cell death executioners (gasdermin D and caspase-7). Cytokines increased insulin release in SC-islets and primary islets, while diminishing insulin secretion in EndoC-{beta}H5 cells. Cytokines reduced glucagon release in SC-islets, which was partially restored by treatment with the incretin hormone glucose-dependent insulinotropic peptide (GIP) with or without a glucagon-like peptide 1 (GLP-1) receptor agonist (liraglutide). ConclusionSC-islets are highly responsive to inflammation with a high degree of similarity to primary islets. Our results support the use of SC-islets as a valid tool in inflammation and diabetes research.

BackgroundHypertension is a highly heritable cardiovascular disorder and a major determinant of cardiometabolic disease, including diabetes. However, the regulatory genes and tissue-specific mechanisms underlying blood pressure variations remain incompletely understood. MethodsLeveraging a well-characterized prospective population-based cohort comprised of 27,308 participants from the Singapore Chinese Health Study (SCHS), we evaluated genome-wide genetic associations for five blood pressure traits: hypertension status, systolic blood pressure, diastolic blood pressure, mean arterial pressure (MAP) and pulse pressure (PP). Additionally, we conducted a transcriptome-wide association study (TWAS), integrating gene expression data from 49 tissues, followed by colocalization and fine-mapping to prioritize regulatory genes. Association of identified variants with incident diabetes was additionally evaluated in the longitudinal data. ResultsWe validated 10 blood pressure loci (P between 1.64 x 10-20 - 4.10 x 10-8) and identified an East-Asian specific splice donor variant at the COL21A1 gene associated with PP (rs149344559, P = 6.78 x 10-10). Integrative analyses prioritized FGF5 in kidney cortex and ENPEP in pituitary tissue as candidate regulatory genes. The blood pressure-lowering allele at ENPEP (T allele, rs1879056) was associated with reduced risk of incident diabetes. Mediation analysis demonstrated that approximately 21% of the genetic association with diabetes was mediated through MAP (Pindirect-effect = 2 x 10-16). ConclusionThis study refines genetic predispositions for blood pressure among East-Asians. We further delineate tissue-specific regulatory pathways underlying blood pressure variations and identify ENPEP-mediated dysfunctions linking blood pressure genetics to diabetes risk, underscoring integrated disease mechanisms.

BackgroundChronic malnutrition in early childhood is a multifactorial condition associated with long-term impairments, yet the physiological and gut microbial pathways underlying differential growth trajectories remain poorly understood. ObjectiveWe aimed to characterize phenotypic growth trajectories and identify the associated gut microbial and body composition signatures in infants during the first year of life. MethodsWe analyzed longitudinal data from birth to 12 months in a South African cohort (Soweto, n=45). Individual linear growth trajectories were modeled using the Jenss-Bayley equation, and children were clustered based on model parameters to identify phenotypic subgroups. Body composition (fat-free mass and fat mass) was measured via deuterium dilution at 6 and 12 months, and gut microbiome development was assessed using 16S rRNA gene amplicons at 4, 6, and 12 months. ResultsWe identified distinct phenotypic subgroups including healthy growth, catch-up growth, and growth faltering, that were obscured at the cohort level. These trajectories diverged most dynamically within the first 6 months of life. Integrated analysis revealed that in the growth faltering cluster, height-for-age and fat-free mass z-scores stabilized between 6 and 12 months, whereas fat mass z-scores (FMZ) declined. This trade-off is consistent with a catabolic state where energy reserves are prioritized for lean tissue and bone growth. Furthermore, at 6 months, the growth faltering cluster was enriched with opportunistic pathobionts (e.g., Paraclostridium). In contrast, the catch-up cluster exhibited a transient enrichment of facultative anaerobes (e.g., Enterobacter), supporting a hypothesis that these oxygen-tolerant taxa may help bridge a transitional microbial state in partially oxygenated or inflamed environments to enable physiological recovery. ConclusionsEarly childhood chronic malnutrition phenotypes in South African infants can be defined by distinct microbial and body composition signatures that diverge within six months of life. Integrated interventions should target both host anabolic state and microbiome transitions to support recovery.